Three trials for Clean up for PCR squared Eluted in Tris HCl in order to get rid of enzymes, dNTPS and any unnecessary contamination, to purify the sample. Analysis: concentrations of these three trials is enough, all around 170ng/uL. Able to prepare and cut pNIC-Bsa4 and
move on to cloning, inserting gene into vector, and further advance in my research.
12042014- Need more analysis of work completed in the lab
Week 11,12,13
1162014- Good Job
Week 8,9,10
Analysis: able to get my primary and secondary on working this confirms elongation of my gene and over replication of it. Will now be moving up to my PCR squared for purification and over amplification.
9232014- Good job
Week 5,6&7
10/3/14 Primary PCR Trial 2
Fig 2. Lane 1 Empty, Lane 2: Kb ladder, Lane 5: my mix (failed nothing shown).
10/2/14 Primary PCR trial1
Fig 1. Lane 1: Empty, Lane 2: Kb ladder, Lane 5: my mix (failed nothing shown), Lane 9: Kb ladder.
Analysis:
Purpose of primary PCR is to put together my oligo mix which partially makes a double strand but gene is not fully assembled. Gene development is verified after running a gel of my pcr mix with a smear shown, but so far i have not been able to get a smear on my gel
9/30/14 Plasmid Midi-Prep
Figure 1. Run1 of Plasmid pNic28-Bsa4 Nano drop spectrophotometry graph, absorbance readings of 1.271. Concentrations is 63.5 ng/uL. Absorbance recorded at 260 nm.
Figure 2. Run2 of Plasmid pNic28-Bsa4 Nano drop spectrophotometry graph, absorbance readings of 1.224. Concentrations is 61.2 ng/uL. Absorbance recorded at 260 nm.
Analysis:
Midi Prep was done for extracting/purifying pNIC BSa4 out of pellet, and remove unwanted cell components.
9/29/14 PCR Primer Design Tails for pNIC-Bsa4 Cloning
Figure2. BsaI's cuts of pNIC28-Bsa4 plasmid.
Figure 3. pNIC28-Bsa4 plasmid digested with BsaI shown is also 1% agarose virtual gel. Lane 1 – 1 kb ladder, lane 2 DNA unmethylated and lane 3 methylated DNA.
Analysis:
pNIC-Bsa4 plasmid was virtually cut with Bsal using NEB cutter. Image of this cut was made and put above as well. Gel of this cut was made too with 1 kb ladder and the gene segments of bsal on the plasmid. The sequence that Bsal cut was removed and my CDS of pF6Fg2 sequence was inserted for cloning.
Week 3&4
9/15/14 PCR
Figure 1. 1000 bp marker to the left, and agarose gel to the right. Lane 2 was 100 bp marker 5ul, lane 3 was Sample A (0.3 ng), lane 4 was Sample B (3 ng), lane 5 was Sample C (30 ng), lane 6 was Sample D (0 ng) nothing, lane 7 100 bp marker.
Analysis: the purpose here for this lab was to amplify purple protein coding sequence in the pGBR22 plasmid using forward and reversed primers. dilutions were made for the tubes. Tube A had a 1:10000, tube B had a 1:1000, tube C had a 1:100. After PCR, gels were made to analyze the samples to see the DNA bands. The gel was run for about 40 mins, at 105 volts until the visible blue marker dye was near the bottom 1/4th of the gel. After gel was run nothing appeared. Unfortunately the gel only showed the two DNA ladders that were placed on lanes 2 and 7. These errors were systematic errors because most of the gel running machines do not work so this being are probable cause of why the gel did not run appropriately. Next possible steps would be to see if one of my lab peers managed to run a gel and get reasonable date out of them so that i can see it and get comparable conclusion for my samples.
9/9/14 RE Digest Day 1
Fig1. Concentration of plasmid pGBR22 using Nanodrop in this case the concentration gather was 265.7 ng/uL
Fig2. All the samples that were used for lanes 4-6 of the gel. from left to right 1st PVUII pGBr22 2nd PVUII & ECORi pGBr22 3rd EcoRi pGBr22.
Fig3. 1 kb DNA ladder key on top and ago rose gel on the bottom; lane 1 of gel skipped, lane 2 1 kb DNA ladder 5 ul, lane 3 had uncut plasmid 10 ng/ul, Lane 4 PVU II pGBr22, Lane 5 PVUII & ECORi pGBr22, Lane 6EcoRi pGBr22. Ran at 100V fro approximately 40-45 mins.
Fig3. 1 kb DNA ladder key on top and ago rose gel on the bottom; lane 1 of gel skipped, lane 2 1 kb DNA ladder 5 ul, lane 3 had uncut plasmid 10 ng/ul, Lane 4 PVU II pGBr22, Lane 5 PVUII & ECORi pGBr22, Lane 6EcoRi pGBr22. Ran at 100V fro approximately 40-45 mins.
Analysis: the purpose for this lab was to make plasmid digestion using EcoRi and PVUII and a combination of both. After the tree samples were prepared, we ran a gel with 1 kbDNA ladder, our three samples and uncut plasmid in a total of five lanes. The gel was run at 100V, until blue dye was half way since DNA id negatively charged for about 40 min. The Bands did appear as i expected even though the lanes were kind of blurry and couldn't tell whether they had other components in them. Next steps would be to analyze the gel with people of greater experience to confirm my gels validity.
Day 2 Transformation 9/8/14
Fig 1. LB culture, 80 ml, bacteria with plasmid DNA pNIC-Bsa4 grown over night for about 16 hrs. in 37 degrees shaker at fast rpm 200-350 rpm.
Day 3 Transformation 9/9/14
Fig 2. Two conical tubes each with LB+Kan pNIC Bsa4 Ecoli #1 weights about 43.58 g and #2 weights about 43.45 g.
Fig 3. Two conical tubes pallets after centrifugation in 4 degrees centrifuge at 6000 x g for 15 mins. Both tubes contain pNIC-Bsa4 Ecoli.
Analysis:
For day 2 and days 3 of transformation bacterial colonies with plasmid pNICBsa4 were overgrown and stored for later usage when we begin Mid Prep. The purpose for day 2 was to grab a bacterial colony and overgrow it over night for about 16 hrs in 37 shaker at fast rpm. Day 3 the contents of the flasks were put into conical tubes were they were spined down in 4 centrifuge at 6000xg for about 15 mins.No contamination in pallets.
982014- Nice work, remember to have all your pictures nexttime
Week 1&2
Figure 1. Culture of Bacterial E.coli DH5alpha with plasmid DNA pNIC-Bsa4 in a 50ul sample.
Figure 1. Culture of Bacterial E.coli DH5alpha with plasmid DNA pNIC-Bsa4 in a 10ul sample.
Analysis: Our transformation of DH5alpha E.Coli did happen and it grew as you can see on the figures above. Whether the plasmid was inserted into the bacteria’s DNA can not be know but there were definitely colonies of bacterial in both agar plates. The next step would be to continue with the lab and over grow the bacteria and see whether the bacteria can expressed our desired protein.
Analysis: The purpose for this lab was to verify DNA purity using nanodrop. Unfortunately we forgot to gather the second graph and were only able to salvage the first one. The recorded concentration was 302.9 ng/ul for the pgbr22 and for the ratios 260/230 and 280/230 the readings were 1.88 and 2.34 respectively, which are close enough to pure pgbr22 which have 1.8 and 2.1 for the ratios 260/230 and 280/230. This validates the sample of pgbr22 and we are able to proced to the DNA sequnceing facility for them to sequence our sample for further use.
Week 14,15
Three trials for Clean up for PCR squared Eluted in Tris HCl in order to get rid of enzymes, dNTPS and any unnecessary contamination, to purify the sample.Analysis: concentrations of these three trials is enough, all around 170ng/uL. Able to prepare and cut pNIC-Bsa4 and
move on to cloning, inserting gene into vector, and further advance in my research.
12042014- Need more analysis of work completed in the lab
Week 11,12,13
1162014- Good Job
Week 8,9,10
Analysis: able to get my primary and secondary on working this confirms elongation of my gene and over replication of it. Will now be moving up to my PCR squared for purification and over amplification.
9232014- Good job
Week 5,6&7
10/3/14 Primary PCR Trial 210/2/14 Primary PCR trial1
Analysis:
Purpose of primary PCR is to put together my oligo mix which partially makes a double strand but gene is not fully assembled. Gene development is verified after running a gel of my pcr mix with a smear shown, but so far i have not been able to get a smear on my gel
9/30/14 Plasmid Midi-Prep
Analysis:
Midi Prep was done for extracting/purifying pNIC BSa4 out of pellet, and remove unwanted cell components.
9/29/14 PCR Primer Design Tails for pNIC-Bsa4 Cloning
Analysis:
pNIC-Bsa4 plasmid was virtually cut with Bsal using NEB cutter. Image of this cut was made and put above as well. Gel of this cut was made too with 1 kb ladder and the gene segments of bsal on the plasmid. The sequence that Bsal cut was removed and my CDS of pF6Fg2 sequence was inserted for cloning.
Week 3&4
9/15/14 PCRAnalysis: the purpose here for this lab was to amplify purple protein coding sequence in the pGBR22 plasmid using forward and reversed primers. dilutions were made for the tubes. Tube A had a 1:10000, tube B had a 1:1000, tube C had a 1:100. After PCR, gels were made to analyze the samples to see the DNA bands. The gel was run for about 40 mins, at 105 volts until the visible blue marker dye was near the bottom 1/4th of the gel. After gel was run nothing appeared. Unfortunately the gel only showed the two DNA ladders that were placed on lanes 2 and 7. These errors were systematic errors because most of the gel running machines do not work so this being are probable cause of why the gel did not run appropriately. Next possible steps would be to see if one of my lab peers managed to run a gel and get reasonable date out of them so that i can see it and get comparable conclusion for my samples.
9/9/14 RE Digest Day 1
Analysis: the purpose for this lab was to make plasmid digestion using EcoRi and PVUII and a combination of both. After the tree samples were prepared, we ran a gel with 1 kbDNA ladder, our three samples and uncut plasmid in a total of five lanes. The gel was run at 100V, until blue dye was half way since DNA id negatively charged for about 40 min. The Bands did appear as i expected even though the lanes were kind of blurry and couldn't tell whether they had other components in them. Next steps would be to analyze the gel with people of greater experience to confirm my gels validity.
Day 2 Transformation 9/8/14
Day 3 Transformation 9/9/14
Analysis:
For day 2 and days 3 of transformation bacterial colonies with plasmid pNICBsa4 were overgrown and stored for later usage when we begin Mid Prep. The purpose for day 2 was to grab a bacterial colony and overgrow it over night for about 16 hrs in 37 shaker at fast rpm. Day 3 the contents of the flasks were put into conical tubes were they were spined down in 4 centrifuge at 6000xg for about 15 mins.No contamination in pallets.
982014- Nice work, remember to have all your pictures next time
Week 1&2
Analysis:
Our transformation of DH5alpha E.Coli did happen and it grew as you can see on the figures above. Whether the plasmid was inserted into the bacteria’s DNA can not be know but there were definitely colonies of bacterial in both agar plates. The next step would be to continue with the lab and over grow the bacteria and see whether the bacteria can expressed our desired protein.
Analysis:
The purpose for this lab was to verify DNA purity using nanodrop. Unfortunately we forgot to gather the second graph and were only able to salvage the first one. The recorded concentration was 302.9 ng/ul for the pgbr22 and for the ratios 260/230 and 280/230 the readings were 1.88 and 2.34 respectively, which are close enough to pure pgbr22 which have 1.8 and 2.1 for the ratios 260/230 and 280/230. This validates the sample of pgbr22 and we are able to proced to the DNA sequnceing facility for them to sequence our sample for further use.